Abstract
BackgroundThe mechanisms by which tumor-specific T cells induce regression of established metastases are not fully characterized. In using the poorly immunogenic B16BL6-D5 (D5) melanoma model we reported that T cell-mediated tumor regression can occur independently of perforin, IFN-γ or the combination of both. Characterization of regressing pulmonary metastases identified macrophages as a major component of the cells infiltrating the tumor after adoptive transfer of effector T cells. This led us to hypothesize that macrophages played a central role in tumor regression following T-cell transfer. Here, we sought to determine the factors responsible for the infiltration of macrophages at the tumor site.MethodsThese studies used the poorly immunogenic D5 melanoma model. Tumor-specific effector T cells, generated from tumor vaccine-draining lymph nodes (TVDLN), were used for adoptive immunotherapy and in vitro analysis of chemokine expression. Cellular infiltrates into pulmonary metastases were determined by immunohistochemistry. Chemokine expression by the D5 melanoma following co-culture with T cells, IFN-γ or TNF-α was determined by RT-PCR and ELISA. Functional activity of chemokines was confirmed using a macrophage migration assay. T cell activation of macrophages to release nitric oxide (NO) was determined using GRIES reagent.ResultsWe observed that tumor-specific T cells with a type 1 cytokine profile also expressed message for and secreted RANTES, MIP-1α and MIP-1β following stimulation with specific tumor. Unexpectedly, D5 melanoma cells cultured with IFN-γ or TNF-α, two type 1 cytokines expressed by therapeutic T cells, secreted Keratinocyte Chemoattractant (KC), MCP-1, IP-10 and RANTES and expressed mRNA for MIG. The chemokines released by T cells and cytokine-stimulated tumor cells were functional and induced migration of the DJ2PM macrophage cell line. Additionally, tumor-specific stimulation of wt or perforin-deficient (PKO) effector T cells induced macrophages to secrete nitric oxide (NO), providing an additional effector mechanism for T cell-mediated tumor regression.ConclusionThese data suggest two possible sources for chemokine secretion that stimulates macrophage recruitment to the site of tumor metastases. Both appear to be initiated by T cell recognition of specific antigen, but one is dependent on the tumor cells to produce the chemokines that recruit macrophages.
Highlights
The mechanisms by which tumor-specific T cells induce regression of established metastases are not fully characterized
Adoptive immunotherapy with wt or perforin knock-out mouse (PKO) effector T cells promotes rapid infiltration of Mac-1+ and GR-1+ cells Lungs from mice bearing 4-day established pulmonary metastases were obtained 24 hours after adoptive transfer of tumor-specific T cells and frozen sections were stained with mAbs against CD4, CD8, NK1.1, Mac-1, and GR-1
As reported by us previously, there were substantial increases in the number of CD4+, CD8+, Mac-1+, and GR-1+ cells in the tumor-bearing lungs of mice that received wt effector T cells (Figure 1) [3]. Comparing these results to those obtained after adoptive transfer of PKO effector T cells there were no apparent differences in the level of infiltrating CD4+, CD8+, Mac-1+, and GR-1+ cells (Figure 1)
Summary
The mechanisms by which tumor-specific T cells induce regression of established metastases are not fully characterized. If TNF was neutralized in T cells deficient in perforin and IFN-γ significant tumor regression was no longer observed [4] These results suggest a triad of molecules that can "compensate" for one another and define a minimal functional requirement for T cells to mediate tumor regression in this model. Immunohistochemical analyses of pulmonary metastases identified macrophages to be a dominant component of the cellular infiltrate following adoptive transfer of therapeutic T cells and IL-2. These same infiltrates were not seen in progressively growing tumors or animals treated with IL-2 alone [2]. Since the CC-chemokines MCP-1 (CCL2), MIP-1α, (CCL3), MIP-1β (CCL4), and RANTES (CCL5), as well as the CXC chemokine IP-10 (CXCL10), and MIG (CXCL9) were found to be associated with a type-1 T cell response [9,10,11], we posited that there may be a link between expression of these chemokines, macrophage infiltration of tumor metastases and therapeutic efficacy
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